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毒害艾美球虫孢子化卵囊cDNA文库的构建
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卞庆松1,韩红玉1,赵其平1,姜连连1,董辉1,闫晓菲1，2,张建哲1，3,黄兵1*
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（1.中国农业科学院 上海兽医研究所 农业部动物寄生虫学重点实验室，上海200232; 2.新疆农业大学
动物医学学院，新疆 乌鲁木齐830052;3.上海师范大学 生命与环境科学学院，上海200234）
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摘要：
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为构建毒害艾美球虫孢子化卵囊cDNA表达文库，用Trizol试剂提取毒害艾美球虫孢子化卵囊总RNA，采用SMART技术，在逆转录酶的作用下将总RNA反转录成第一链cDNA，经LD-PCR扩增合成双链cDNA（ds cDNA）。经蛋白酶K消化、SfiⅠ酶切，过CHROMA SPIN-400TM柱去除小于400bp的片段，与λTriplEx2TM噬菌体载体连接，用GigapackⅢ GoldTM载体蛋白包装，构建了cDNA噬菌体表达文库。经测定，原始文库容量为4.72×106pfu/mL，扩增后的文库容量为2.62×1010 pfu/mL，重组率为975%，插入片段为500～1100bp。证实，该文库质量良好，为毒害艾美球虫新基因的筛选、克隆及功能研究奠定了基础。 |
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关键词：
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球虫；毒害艾美球虫；孢子化卵囊；cDNA文库；SMART技术 |
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中图分类号：S 852.723：Q 81 文献标识码：
A 文章编号：1673-4696(2008)03-0196-04 |
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Construction of a cDNA library for Eimeria necatrix sporulated oocysts |
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BIAN Qing-song1,HAN Hong-yu1,ZHAO
Qi-ping1,JIANG Lian-lian1,DONG Hui1,
YAN Xiao-fei1，2,ZHANG Jian-zhe 1,3,HUANG Bing1 |
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(1.Key Laboratory of Animal Parasitology of the Ministry of Agriculture/ Shanghai Veterinary Research Institute，
Chinese Academy of Agricultural Sciences，Shanghai 200232,China;2.College of Veterinary Medicine，Xinjiang
Agricultural University，rümqi 830052,China;3.College of Life and Environment Sciences,Shanghai Normal
University,Shanghai 200234,China) |
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Abstract: To construct a cDNA expression library for Eimeria necatrix sporulated oocysts,the total RNA of the sporulated oocysts of E.necatrix was extracted using Trizol reagent and then was reverse-transcripted with reverse-transcriptase by SMART (switching mechanism at 5′ end of RNA transcript) technique.The double-strand cDNA(ds cDNA) was synthesized with the first strand cDNA as template by amplification using long-distance PCR(LD-PCR).cDNA fragments less than 400bp fragments were removed by CHROMA SPIN-400TM columns after the ds cDNA was digested with proteinase K and SfiⅠ，and the purified fragments were ligated into λTriplEx2TM vector and packaged with the GigapackⅢ GoldTM.The results showed that the capacity of the un-amplified library was 472×106 pfu/mL，the capacity of the amplified library was 2.62×1010 pfu/mL，the library recombination rate was 97.5%,and the inserted cDNA fragments were 500-1100bp in length.These results revealed that the constructed cDNA expression library for E.necatrix sporulated oocysts had good quality which provided a basis for screening,cloning and further studies of new genes of E.necatrix. |
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Key words: chicken；coccidia；Eimeria necatrix；sporulated oocysts；cDNA library；SMART technique |
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